Glidden grew up on his father's farms in New Hampshire and (later) New York State. In 1837 he married Clarissa Foster, and the couple bought a farm in De Kalb, Illinois. Glidden's two sons with Clarissa later died in an epidemic, and Clarissa herself died in childbirth. In 1851 Glidden married Lucinda Warne.

In 1873 Glidden saw an ineffective example of barbed wire, a “thorn hedge,” designed to keep cattle from trampling crops. Glidden began experimenting with ways to improved barbed wire, and the next year he received a patent for a machine that added the barbs to the wire mechanically, thus allowing for mass production. Glidden and his partner, Isaac L. Ellwood, established the Barb Fence Company in De Kalb, and in a few years, barbed wire was being used all over the West. Barbed wire accelerated the development of the region, closed the open range, and ended the golden age of the cowboy, who had roamed freely over wide stretches of land. Glidden was soon one of the richest men in America. He invested in the fancy Glidden Hotel in De Kalb and bought 180,000 acres of land in Texas, where he raised 15,000 head of cattle.